Method for synthesizing short single strand deoxyribonucleotide probe

Abstract

The invention belongs to the field of DNA (deoxyribonucleic acid) biosynthesis, and in particular relates to a method for synthesizing a short single strand deoxyribonucleotide probe and application thereof. The short single strand deoxyribonucleotide probe can be used for detecting small non-coding ribonucleic acid (RNA) such as micro ribonucleic acid. The method comprises the following steps of: (1) preparing plasmids recombined with template DNA; (2) cutting and connecting the template DNA, performing enzyme digestion on the recombined plasmids to obtain the template DNA, and connecting the template DNA end to end to form a ring; (3) cutting a single strand, performing rolling-circle replication, cutting one strand of the template DNA by using nickase, adding polymerase to perform rolling-circle replication by taking annular DNA with a nick as a template, and amplifying to obtain a DNA single strand containing a neck ring-probe structure; and (4) preparing target short single strand DNA, cutting the product of the step (3) by using a type II incision enzyme to form a target single strand DNA probe and a byproduct DNA, and separating and purifying through PAGE (polyacylamide gel electrophoresis) to obtain a single strand DNA probe without mutation. The short single strand deoxyribonucleotide probe has wide applications in the fields of detection of small non-coding RNA and diagnosis and treatment of small non-coding RNA related diseases.

Description

technical field [0001] The invention belongs to the field of DNA biosynthesis, in particular to a method for synthesizing a short single strand deoxyribonucleotide (short single strand DNA, sssDNA) probe, which can be used to detect small non-coding ribonucleic acid (small non-coding RNAs) , such as microRNA (microRNA). Background technique [0002] Short single-strand deoxyribonucleotides (sssDNA) can be used as probes for scientific research and medical diagnosis. In the past decade or so, the research on small non-coding RNAs, especially microRNAs, has been very hot. Scientists and pharmaceutical companies are also very optimistic about the prospect of using microRNAs as targets to develop diagnostic kits and drugs. Since microRNAs usually have only 18-30 bases (Bartel, 2004), and the difference between a family of microRNAs may be only 1-2 bases (www.mirbase.com), if you use the hybridization method to detect microRNAs, The purity requirements for probes are high. If ...

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Problems solved by technology

PCR (polymerase chain reaction, polymerase chain reaction) is used to produce short nucleotide probes with a length of 30 bases (Bertilsson et al., 2002, Appl Environ Microbiol), but this method has two disadvantages : One is that PCR needs to amplify with chemically synthesized primers, and the synthetic primers always have mutations, which makes the PCR products carry the mutations in the primers, causing the products to be less than 100% pure, and to The length of the amplified product is limited; another disadvantage is that the product synthesized by PCR is double-stranded, and additional steps are required to separate the double-stranded and purify the target single-stranded

This method has great limitations: constant temperature amplification has strict requirements on the length of the amplified oligonucleotide, if the oligonucleotide to be amplified is too long, the Tm value will be too high, and the incision will end The oligonucleotide fragment produced after the enzyme acts on the double-stranded oligonucleotide fragment will not fall off from the template; on the contrary, if the oligonucleotide to be amplified is too short, the Tm value will be too low, and the oligonucleotide Acid fragments are easily detached from the template

The disadvantages of this method: one is that the errors in the chemically synthesized template and primers are brought into the product; the other is that there is no way to cut the long single strand into specific short strands; Dicerase cuts, but requires the target DNA fragment to be replicated and amplified to contain a restriction site, that is, it cannot amplify a specific sequence without endonuclease, and the cut product is still double-stranded, which cannot be used for single-strand detection. Needle experiments and diagnostics

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